Plant biomass is a carbon-neutral renewable resource and biomass conversion, particularly cellulosic feedstock conversion, is receiving much attention as a cleaner alternative to oil and as a result of its abundance and relatively low cost. The current process of converting starch to bioethanol is well established, but the energy cost is high and, accordingly, the need to develop a more feasible process is evident.
The industrial process of converting starch to bioethanol generally involves four steps which include: (i) extraction of starch from the biomass, (ii) the conversion of the starch to yield fermentable sugars, which are then (iii) fermented to ethanol upon the addition of yeast. In the final step (iv) ethanol is refined and concentrated by distillation. Extraction of starch is accomplished via wet milling or dry milling. Starch may be converted to fermentable sugars via acid hydrolysis or enzymatic hydrolysis (enzyme hydrolysis is now generally used). Enzymatic hydrolysis is initiated when starch is pre-treated to yield a viscous slurry, which is then liquefied by heat treatment and alpha-amylase. The starch is cooked and undergoes saccharification after addition of glucoamylase. Yeast is added after cooling the mixture for fermentation of sugars to ethanol. The process includes large temperature changes (typically in the range of about 32-120° C.) using vast amounts of heating energy which significantly adds to the cost of the process. With the intention to increase net energy yield, the hydrolysis temperature required to generate glucose could be lowered to that of the fermentation step, therefore carrying out saccharification and fermentation simultaneously (SSF). Research groups currently focus on either improving the commercial hydrolysing enzymes applied in the process, or improving microbes producing the hydrolytic enzymes necessary for the process to proceed efficiently.
A raw starch hydrolyzing (RSH) enzyme cocktail, Stargen 001 (Genencor) has been developed, which converts starch into dextrins at low temperatures (<48° C.) and hydrolyses dextrins into sugars during SSF. The cocktail contains an acid-stable alpha-amylase from Aspergillus kawachi and glucoamylase from Aspergillus niger. Using the RSH enzyme saves heating energy as jet cooking is eliminated and less water and fewer chemicals are needed for the process.
To eliminate commercial enzyme purchase costs, SSF has been performed effectively with mixed cultures, where one organism is amylolytic, and the other responsible for ethanol production. The amylolytic organism acts as the saccharifying agent, therefore replacing the addition of commercial saccharifying enzymes. However, in these systems the amylolytic organism utilises most of the soluble starch for growth, which leaves little sugars for the fermentative organism to convert to ethanol.
Generating an amylolytic fermentative organism may address this shortcoming. A more cost-effective procedure where an organism produces sufficient amounts of amylolytic enzymes to sustain growth on raw unmodified starch as sole carbon source for the production of ethanol as product is depicted in FIG. 2. Applying a raw starch utilising yeast in the starch conversion process will have all the benefits from an SSF procedure, such as a lowered heating energy requirement and chemical usage. The added benefit will be elimination of the large cost associated with commercial enzyme purchase. The engineered organism producing amylolytic enzymes and ethanol would be suitable for a Consolidated Bioprocessing (CBP) process. In the long term, generation of ethanol and coproducts employing a CBP process will ensure the production of commodity chemicals and animal feeds in a sustainable manner in a biorefinery environment.
Genetic engineering is used extensively for producing hosts with desired characteristics for the starch industry. Mainly alpha-amylases and glucoamylases are expressed in heterologous hosts to ensure higher enzyme productivity compared to the native host. Expression of thermostable enzymes as well as the ability to produce more than one desirable enzyme in one host enables the generation of more competitive organisms for the industry.
Yeasts displaying glucoamylases (e.g. Kondo et al., 2002) and alpha-amylases have been previously created (Shigechi et al., 2002). However, engineering a host strain to express raw starch hydrolysing enzymes would be even more advantageous. Very few groups have reported results on yeast strains which are able to utilise raw starch as carbon source with initial approaches utilising the Rhizopus oryzae glucoamylase, which is secreted or displayed on the surface of the yeast (Ashikari et al., 1989; Murai et al., 1998). Several different strains have been created where the alpha-amylases from Streptococcus bovis has been combined with the glucoamylase in the hope to better the amylolytic activity and therefore the ethanol productivity of the generated strains (Khaw et al., 2006; Shigechi et al., 2004b). An alpha-amylase preparation from B. licheniformis (Murai et al., 1998) has also been used the source of alpha-amylase activity.
Kim J H et al. (2010) Biotechnol Lett. February 4, developed a strain of S. cerevisiae that produces ethanol directly from starch. Two integrative vectors were constructed to allow the simultaneous multiple integration of the Aspergillus awamori glucoamylase gene (GA1) and the Debaryomyces occidentalis alpha-amylase gene (AMY) and glucoamylase with debranching activity gene (GAM1) into the chromosomes of an industrial strain of S. cerevisiae. Yamakawa et al. (2010) Appl. Microbiol. Biotechnol. February 24. [Epub ahead of print] demonstrated batch ethanol fermentation from raw using a yeast diploid strain coexpressing the maltose transporter AGT1, alpha-amylase, and glucoamylase.
The present invention aims to provide a beneficial enzyme combination for the hydrolysis of starch for use in alcohol production processes. The invention also describes beneficial yeast strains which may find particular utility in fermentation processes and which may also be engineered to express an enzyme combination of the invention.